Stenotrophomonas maltophilia (Sm) is an environmental, Gram-negative bacterium that has recently emerged as an important opportunistic and nosocomial pathogen, particularly of the respiratory tract and bloodstream. Infection with S. maltophilia is often very difficult to treat because of the resistance that S. maltophilia strains have toward multiple types of antibiotics. Despite the role of Sm in pneumonia, bloodstream infections, and various other diseases, including urinary tract, CNS, eye, skin, and soft tissue infections, the pathogenesis of Sm has been grossly understudied. With the aid of a small NIH R03 grant, we previously embarked upon research aimed at developing methods to study Sm. After having established a murine model for Sm pneumonia and developed means for mutagenizing the sequenced Sm strain K279a, we investigated the functioning of type II protein secretion (T2S) in Sm. Previous work from our lab and others had shown that T2S systems are major facilitators of virulence in various pathogens, including other agents of pneumonia and bloodstream infection. Proteins secreted via T2S typically include a wide variety of degradative and tissue- destructive enzymes. We documented that the Xps T2S system is functional in strain K279a, secreting at least seven protein species. Importantly, we determined that Xps mediates the export of two serine proteases (StmPr1 and StmPr2) that cause a human lung epithelial cell line (i.e., type I A549 cells) to undergo rounding, actin-rearrangement, detachment, and death. Thus, we hypothesize that Xps T2S and its substrates, including StmPr1 and StmPr2, are critical in the pathogenesis of S. maltophilia. Genomic data further indicate that Sm possesses a second T2S system (Gsp) as well as type IV (T4S) and type I (T1S) protein secretion systems. We hypothesize that proteins secreted by these other systems also represent virulence factors of Sm. To address these hypotheses, we propose to i) test the Xps mutant in two different murine models of disease, the published pneumonia model and a new bacteremia model (Aim 1), ii) test a stmPr1 stmPr2 mutant in the pneumonia and bacteremia models, and with confirmation of the importance of StmPr1 and/or StmPr2 begin to determine how the proteases mediate host cell death (Aim 2), and iii) test Gsp, T4S, and T1S mutants in the two disease models. From the results of this exploratory R21 grant, we will establish a working foundation for the nascent Sm pathogenesis field such that future efforts will be able to focus on characterizing the secretion system(s) and secreted proteins that are most important. In addition to providing much-needed insight into Sm pathogenesis, the data obtained have the potential to both uncover new types of virulence determinants and lead to new forms of disease diagnosis, treatment, or prevention. .